Molecular and Cell Biology Laboratory, Dulbecco Center for Cancer Research, La Jolla, California, United States of America.
PLoS One. 2010 Feb 12;5(2):e9197. doi: 10.1371/journal.pone.0009197.
The appropriate control of mitotic entry and exit is reliant on a series of interlocking signaling events that coordinately drive the biological processes required for accurate cell division. Overlaid onto these signals that promote orchestrated cell division are checkpoints that ensure appropriate mitotic spindle formation, a lack of DNA damage, kinetochore attachment, and that each daughter cell has the appropriate complement of DNA. We recently discovered that AMP-activated protein kinase (AMPK) modulates the G2/M phase of cell cycle progression in part through its suppression of mammalian target of rapamycin (mTOR) signaling. AMPK directly phosphorylates the critical mTOR binding partner raptor inhibiting mTORC1 (mTOR-raptor rapamycin sensitive mTOR kinase complex 1). As mTOR has been previously tied to mitotic control, we examined further how raptor may contribute to this process.
METHODOLOGY/PRINCIPAL FINDINGS: We have discovered that raptor becomes highly phosphorylated in cells in mitosis. Utilizing tandem mass spectrometry, we identified a number of novel phosphorylation sites in raptor, and using phospho-specific antibodies demonstrated that raptor becomes phosphorylated on phospho-serine/threonine-proline sites in mitosis. A combination of site-directed mutagenesis in a tagged raptor cDNA and analysis with a series of new phospho-specific antibodies generated against different sites in raptor revealed that Serine 696 and Threonine 706 represent two key sites in raptor phosphorylated in mitosis. We demonstrate that the mitotic cyclin-dependent kinase cdc2/CDK1 is the kinase responsible for phosphorylating these sites, and its mitotic partner Cyclin B efficiently coimmunoprecipitates with raptor in mitotic cells.
CONCLUSIONS/SIGNIFICANCE: This study demonstrates that the key mTOR binding partner raptor is directly phosphorylated during mitosis by cdc2. This reinforces previous studies suggesting that mTOR activity is highly regulated and important for mitotic progression, and points to a direct modulation of the mTORC1 complex during mitosis.
有丝分裂的进入和退出的适当控制依赖于一系列相互关联的信号事件，这些信号事件协调驱动细胞准确分裂所需的生物学过程。在这些促进协调细胞分裂的信号之上，存在着确保有丝分裂纺锤体形成、没有 DNA 损伤、动粒附着以及每个子细胞都具有适当的 DNA 数量的检查点。我们最近发现，AMP 激活的蛋白激酶（AMPK）通过抑制哺乳动物雷帕霉素靶蛋白（mTOR）信号来调节细胞周期 G2/M 期的进展。AMPK 直接磷酸化关键的 mTOR 结合伴侣雷帕霉素敏感的 mTOR 激酶复合物 1（mTOR-raptor rapamycin sensitive mTOR kinase complex 1，mTORC1）的 Raptor，从而抑制 mTORC1。由于 mTOR 先前与有丝分裂控制有关，我们进一步研究了 Raptor 如何促进这一过程。
方法/主要发现：我们发现 Raptor 在有丝分裂的细胞中高度磷酸化。利用串联质谱法，我们在 Raptor 中鉴定了多个新的磷酸化位点，并使用磷酸化特异性抗体证明 Raptor 在有丝分裂中磷酸化在磷酸丝氨酸/苏氨酸-脯氨酸位点上。在标记的 Raptor cDNA 中进行定点突变，并使用一系列针对 Raptor 不同位点生成的新磷酸化特异性抗体进行分析，揭示了 Raptor 中 Ser696 和 Thr706 两个关键位点在有丝分裂中被磷酸化。我们证明有丝分裂周期蛋白依赖性激酶 cdc2/CDK1 是负责磷酸化这些位点的激酶，其有丝分裂伴侣 Cyclin B 在有丝分裂细胞中与 Raptor 有效地共免疫沉淀。
结论/意义：这项研究表明，关键的 mTOR 结合伴侣 Raptor 在有丝分裂过程中被 cdc2 直接磷酸化。这强化了先前的研究结果，表明 mTOR 活性受到高度调节，对有丝分裂进程很重要，并指出在有丝分裂过程中对 mTORC1 复合物进行直接调节。
